B And T Lymphocyte Attenuator Expression Research Articles

Targeting BTLA with the peptide inhibitor HVEM(14-39) – A new way to restore the activity of T cells in melanoma

The complex of B- and T-lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) plays a critical role in immune regulation and has emerged as a promising therapeutic target for cancer treatment. In this study, we investigated the potential of the peptide inhibitor HVEM(14-39) to restore peripheral T cell activity in patients with advanced melanoma. In these patients, CD8+ T cells downregulated BTLA expression and increased HVEM expression upon activation. The addition of HVEM(14-39) reduced the percentage of BTLA+ CD8+ T cells and increased the subpopulation of HVEM+ CD8+ T cells. Additionally, HVEM(14-39) enhanced T cell activation, proliferation, and the shift toward effector memory T cell subpopulations. Finally, this peptide affected the proliferation rate and late apoptosis of melanoma cell line in co-culture with T cells. These findings suggest that HVEM(14-39) can overcome T cell exhaustion and improve antitumor responses. Peptide-based immunotherapy targeting the BTLA-HVEM complex offers a promising alternative to monoclonal antibody-based therapies, with the potential for fewer side effects and higher treatment efficacy.

Modulation of the Btla-HVEM Axis to Enhance CAR T Cell Immunotherapy Against Cancer

Introduction: The efficacy of adoptive T cell immunotherapies against cancer, such as chimeric antigen receptor (CAR) T cells, is severely blunted by the immunosuppressive tumor microenvironment (TME). We sought to investigate the role of the TME in cancer resistance to effector T cells in order to define actionable targets to enhance CAR T cell immunotherapies. We initially used Hodgkin lymphoma (HL) as an ideal tumor model since it is characterized by a TME that is profoundly infiltrated by immunosuppressive cells, and then we expanded our findings to multiple cancer models. We first sought to identify the dominant interactions of immunosuppressive cellular compartments and effector T cells by analyzing single-cell RNA sequencing data on a total of 26 (4 exploratory + 22 validation) HL patient tumor biopsies. Using the CellPhoneDB algorithm, we inferred that the ligand B- and T-lymphocyte attenuator (BTLA) on effector T cells and the receptor Herpesvirus entry mediator (HVEM, TNFRSF14) on immunosuppressive cells (e.g., regulatory T cells, monocytes) strongly interact in the TME and promote T cell dysfunction ( Fig 1a). Akin to the canonical checkpoint PD1, BTLA recruits two potent tyrosine phosphatases, SHP-1 and SHP-2, to disable early T cell activation. Thus, we rationalized that BTLA expression on T cells might reduce their anti-tumor function. We hypothesized that deleting BTLA in CAR T cells would abolish BTLA-HVEM trans interactions at the immunological synapse and unleash the cytotoxic potential of CAR T cells. Methods and Results: We first generated BTLA KO anti-CD30 CAR T (CART30) cells against HL. To test their function in vivo, 15x10 6 HDLM-2 (CD30+HVEM+ HL) cells were subcutaneously implanted into NSG mice on day 0, and on day 62, 3x10 5 CAR30+ T cells were infused intravenously. Our results demonstrated that BTLA KO significantly enhances the function of anti-CD30 CAR T cells in HVEM+ HL ( Fig 1b), as assessed via tumor size (caliper) and CART30 expansion in the peripheral blood (flow cytometry). Additionally, we generated BTLA KO 4-1BBζ CART19 cells, which showed greater in vivo anti-tumor function in a subcutaneous tumor model of DLBCL (CD19+ HVEM+ OCI-Ly18). Serum collected from OCI-Ly18-bearing NSG mice infused with BTLA KO CART19 was enriched in effector cytokines (e.g., TNF, IFNγ, IL-2) as measured by Luminex. We then extended these findings into HVEM+ solid tumor models. BTLA KO improved tumor control in vitro in short-term killing experiments (Incucyte SX5) for both CAR and TCR T cells, respectively directed against prostate cancer (HER2+ PC-3) and melanoma (GP-100+ DM-6). Importantly, we demonstrated that BTLA KO in primary BALB/c-derived murine CAR T cells enhances tumor control in A20 murine lymphoma. In this model, BTLA KO tumor-infiltrating CAR T cells showed substantially reduced exhaustion in the TME relative to wild-type CAR T cells. Mechanistically, we showed that targeted mutations in BTLA intracellular tyrosine motifs effectively reduce the recruitment of SHP-1/2 and preserve the docking domain of the pro-stimulatory Grb2, thus maintaining high NFAT signaling and increased persistence in vivo. These effects were dependent on the expression of HVEM on tumor cells. Finally, we found that high BTLA RNA expression in tisagenlecleucel infusion products correlates with poor response to treatment in patients with DLBCL and FL (NCT02030834). Conclusion: Our results reveal a critical role of the BTLA-HVEM axis in inhibiting CAR T cell function, and demonstrate that CRISPR-Cas9 deletion of BTLA leads to enhanced anti-tumor efficacy in multiple models of cancer. The key mechanism is the reduction of SHP-1/2 recruitment and the consequent increase in CAR T cell activation. The results of this study will be translated into a first in human clinical trial of BTLA-deficient CAR T cells for relapsed or refractory cancer.

Vactosertib, a Novel TGFb Type I Receptor Kinase Inhibitor, Improves T-Cell Fitness:a Single-Arm, Phase 1b Trial in Relapsed/Refractory Multiple Myeloma

The prognosis for Multiple Myeloma (MM) patients (pts) has improved substantially over the past two decades with the success of immunomodulatory drugs (IMiDs), monoclonal antibodies, chimeric antigen receptor (CAR) T-cells and bispecific T-cell engagers (BiTEs) that broke the circulus vitiosus of tumor-induced immunosuppression to reengage the immune system. Functional blockade of the transforming growth factor-beta (TGF) signaling pathway improves the efficacy of cytotoxic and immune therapies, therefore we investigator the impact of TGF-inhibition in an steroid free regimn. Methods: We conducted a phase 1b study to determine the safety, efficacy, and maximal tolerated dose (200 mg po bid) of the potent, orally-available TGF type I receptor kinase inhibitor vactosertib in relapsed and/or refractory multiple myeloma (RRMM) pts who had received -2 lines of chemoimmunotherapy in combination with standard dosage of Pomalidomide without any corticosteroids. Results: Twenty one pts were enrolled in the phase 1b dose escalation cohort and received vactosertib plus pomalidomide as indicated (Fig. 1A). Pts' characteristics are described in Table 1The summary of grade 1-4 adverse events is shown (Table 2). A single grade 4 elevated bilirubin (5%) and a single grade 4 elevated lipase (5%) were observed as well as four grade 4 events of decreased neutrophils (19%). Four pts demonstrated a PR, two had a MR, nine had SD and four had disease progression (Fig. 1B). The median time to response for all pts and the responders eight weeks and median duration of response was ~12 weeks. The best overall response for individual pts to treatment is shown (Fig. 1C). Clinical activity was observed across all four dose levels (Table 3). To probe the tumor intrinsic anti-myeloma activity of vactosertib, we first determined the relative effect of vactosertib compared to the IMiDs pomalidomide and lenalidomide (Fig. 2A, B). The effect of vactosertib was greater than that observed with either lenalidomide or pomalidomide. MMCLs and patient CD138 + cells were then treated with vactosertib in combination with pomalidomide, and importantly, vactosertib with pomalidomide synergistically reduced myeloma viability (Fig. 3A). We next probed the systemic, tumor extrinsic effects of vactosertib by analyzing cytokines immune and tumor cells present within patient BM samples. (Fig. 4A). (Fig. 4B). Therefore, we explored the immunophenotype of CD8 + T-cells isolated from patient BM prior to and at the EOT. The relative percentage of T-cells that expressed the immunosuppressive markers programmed cell death-1 (PD-1), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), B and T lymphocyte attenuator (BTLA), and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) was reduced following treatment relative to their expression prior to treatment (Fig. 4C). Results indicated that vactosertib and pomalidomide treatment in vivo reduced PD-1, TIM3, BTLA and CTLA-4 expression on patient CD8 + T-cells. Next, CD8 + T-cells were isolated from patient peripheral blood and cultured in the presence of TGF-1, vactosertib, pomalidomide, or vactosertib plus pomalidomide (Fig. 5B). Treatment of patient CD8 + T-cells with vactosertib decreased PD-1 expression up to 40% and decreased TIM3 expression up to 65%. Pomalidomide alone did not significantly reduce PD-1, TIM3, BTLA, or LAG-3 on patient CD8 + T-cells. Vactosertib and pomalidomide also reduced TIM3 levels on CD8 + T-cells from most pts. (Fig. 5C, D). (Fig. 5 G, H). Results indicated that vactosertib or pomalidomide did not increase CD138 + cell death (Fig. 5I). Conclusion: Vactosertib combined with pomalidomide was well-tolerated at all doses, had a manageable adverse event profile and induced durable responses with 80% progression-free survival (PFS-6) at 6 months, Vactosertib reduced TGF- in patient bone marrow and suppressed PD-1 expression on CD8 + T-cells and lead to reduction of PD-L1/PD-L2 expression on CD138 + cells and enhanced autologous T-cell cytotoxicity. Taken together, our results support the safety and efficacy of vactosertib to treat RRMM and revealed that vactosertib modulates the T-cell immunophenotype and reinvigorates T-cell fitness.

Dual inhibition of BTLA and PD-1 can enhance therapeutic efficacy of paclitaxel on intraperitoneally disseminated tumors

BackgroundExpression of immune checkpoints in the tumor microenvironment is one mechanism underlying paclitaxel (PTX) chemoresistance. This study aimed to investigate whether the addition of checkpoint blockade to PTX can improve...

BTLA transcriptomic expression correlates with high levels of PD-1, PD-L2, and CTLA-4 checkpoints and with its ligand, HVEM, across diverse solid cancers.

e14641 Background: B and T lymphocyte attenuator (BTLA) is an immune checkpoint that is highly expressed on tumor-specific T cells and has been shown to be upregulated in several cancer types. The binding of BTLA to its ligand herpesvirus entry mediator (HVEM, also known as TNFRSF14) inhibits activation and proliferation of T lymphocytes, leading to decreased tumor killing. Early clinical trials of BTLA inhibitors are ongoing with preliminary results showing modest effects in advanced solid tumors. More recent clinical data suggests that combination checkpoint therapy with anti-BTLA and anti-PD-1 treatment provides greater benefit compared to monotherapy in patients with lymphoma. Methods: We analyzed RNA expression levels of 397 genes in various solid tumor types from 514 patients with various types of solid tumors seen at University of San Diego (UCSD), Moores Cancer Center. RNA expression was quantified at a CLIA-approved laboratory Omniseq (https://www.omniseq.com). Transcript abundance was normalized to internal housekeeping gene profiles and ranked (0-100 percentile) in a standardized manner to a reference population of 735 tumors spanning 35 histologies. We analyzed tumors with “High” (75-100 percentile rank) BTLA expression profiles. Odds ratio for high BTLA expression was calculated and multivariate analysis was utilized to determine significant correlations for multiple genes. Results: A total of 96 (18.7%) tumors had high BTLA (>75) expression across 32 different histologies. Cancers with the highest proportion of BTLA expression were small intestine (42%), breast (31%), pancreatic (29%), stomach (24%), neuroendocrine (20%), lung (20%), head and neck (17%) and colorectal (13%). There was significant association for BTLA and high PD-1 (p < 0.001), high CTLA-4 (p < 0.001), high PD-L2 (p = 0.05) and its ligand HVEM (p < 0.01). There was no significant association between BTLA expression and PD-L1, gender or stomach, neuroendocrine, head and neck, and lung histologies. Conclusions: High BTLA expression was found in a large number of tumor samples and significantly associated with other immune checkpoints with FDA-approved drugs. Furthermore, HVEM expression (ligand for BTLA) significantly correlates with high BTLA transcriptome levels. The BTLA/HVEM pathway has been associated with suppression of T cell-mediated anti-tumor activity and resistance to anti-PD-1 immunotherapy leading to poor clinical outcomes. The utilization of combination checkpoint therapy involving BTLA inhibitors and specific checkpoint blockade is currently being explored. Our results suggest that patient selection based on the RNA expression level of immune markers matched to corresponding immunotherapy agent, rather than a population-based approach, may be ideal for optimizing treatment response.

A pan-cancer analysis of the biological function and clinical value of BTLA in tumors

B and T-lymphocyte attenuator (BTLA) plays an immunosuppressive role by inhibiting T- and B-cell functions. BTLA is associated with a variety of diseases, especially cancer immunity. However, the function of BTLA in various cancers and its clinical prognostic value have still not been comprehensively analyzed. This study aimed to identify the relationship between BTLA and cancer from the perspectives of differences in BTLA expression, its clinical value, immune infiltration, and the correlation with immune-related genes in various cancers. Data regarding mRNA expression, miRNA expression, lncRNA expression, and clinical data of patients of 33 existing cancers were collected from the TCGA database. Target miRNA of BTLA and the lncRNA that interacts with the target miRNA were obtained from the StarBase database. Based on bioinformatics analysis methods, the relationship between various types of cancers and BTLA was thoroughly investigated, and a competing endogenous RNA network of BTLA, target miRNA, and interacting lncRNA was constructed. The Kaplan-Meier (KM) prognostic analysis of BTLA and target miRNA (has-miR-137) in various types of cancers was completed using the KM plotter. BTLA expression varied in different cancers, with statistical significance in nine cancer types. KM plotter to analyze the overall survival (OS) and regression-free survival prognosis of cancer patients revealed that the BTLA expression was statistically different in the OS of 11 types of cancers out of 21 types of cancers; the OS of 8 type of cancers was also statistically different. Correlation analysis of tumor immune genes revealed a positive correlation of BTLA expression with immunosuppressive gene (CTLA4 and PDCD1) expression. Gene Set Enrichment Analysis showed that BTLA and its co-expressed genes mainly act through biological processes and pathways, including immune response regulation, cell surface receptor signaling pathway, antigen binding, antigen receptor-mediated signaling pathway, and leukocyte migration. BTLA has the potential as a prognostic marker for CLL, COAD, NSCLC, and OV and a diagnostic marker for CLL, COAD, and KIRC. BTLA has a close and complex relationship with the occurrence and development of tumors, and cancer immunotherapy for BTLA is worthy of further analysis.

B and T lymphocyte attenuator as a C-reactive protein and IgA associated auxiliary diagnostic marker for pulmonary tuberculosis: a case-control study.

Screening and identification of hematologic molecular indicators of pulmonary tuberculosis (PTB) is crucial for its diagnose and therapy. Therefore, our work aims to detect the diagnostic value of blood marker B and T lymphocyte attenuator (BTLA) in PTB, and provide a certain theoretical basis for the auxiliary diagnosis of PTB. Based on the inclusion criteria, 56 Patients with clinically confirmed pulmonary TB by clinical between January 2020 and December 2021 at our hospital were selected as the research objects of this study. Fifty-two matched healthy population at our hospital was used as the control group. Clinical characteristics were got from clinical laboratory. Real-time polymerase chain reaction (RT-PCR) was used to analyze changes in BTLA along with its ligand in peripheral blood. Changes in BTLA on the surface of different cells were analyzed by flow cytometry. The correlation test was used to determine the associations between BTLA and clinical indicators. Receiver operating characteristic (ROC) curve analysis was used to evaluate the auxiliary diagnostic value in PTB of BTLA expression from different sources. Compared with the control, changes in peripheral blood BTLA in the PTB group were significantly increased (P=0.0187) rather than its ligand. Changes in BTLA on the surface of CD68 and antigen-presenting cell (APC) CD11c were significantly increased in the PTB group (P=0.0004, P<0.0001), while changes in BTLA on the surface of CD4+ T and CD8+ T cells were not significantly different (P=0.0792, P=0.8706). The expression of BTLA+CD11c+ was negatively correlated with the expression of immunoglobulin A (IgA) (r=-0.2934, P=0.0282) and positively related to C-reactive protein (r=0.3277, P=0.0137). ROC curve analysis suggested that the area under the curve (AUC), sensitivity and specificity of BTLA RT-PCR detection were 0.6315, 53.57%, 57.69% while for BTLA+CD11c+ detection were 0.8039, 88.46% and 73.21% and for BTLA+CD68+ detection were 0.6973, 60.71% and 61.54%. BTLA is highly expressed in peripheral blood and specific cell types of patients with PTB and is correlated with specific clinical indicators, which may be an important molecular marker for the auxiliary diagnosis of PTB.

PDGFR kinase inhibitor protects against septic death via regulation of BTLA.

Sepsis, defined as life-threatening organ failure caused by a dysregulated host response to severe infection, is a major cause of death among intensive care unit patients. Therapies targeting on immunomodulatory is a new research field in sepsis treatment. B- and T-lymphocyte attenuator (BTLA) is an inhibitory costimulatory factor molecule of B and T lymphocytes. Studies have shown that elevated expression of BTLA in lymphocytes can reduce mortality in sepsis, but its regulatory compounds and the underlying mechanism remains to be elucidated. Here, we show that treatment with CP-673451 significantly decreases mortality of septic mouse. CP-673451 is a PDGFR kinase inhibitor which can promote the expression of BTLA, inhibit the release of chemokines such as CXCL13, and reduce first the chemotaxis of B cells to the peripheral blood and vital organs. CP-673451 also inhibits both the release of cytokines and chemokines such as IL-1β, IL-6, IL-10, TNF-α, CCL1, CCL2 and CCL7 and reduces both the chemotactic ability of T cells. This suggests that CP-673451 may prevent septic death by inhibiting lymphocyte chemotaxis and alleviating “cytokine storm”. In conclusion, our study provides a new therapeutic target and an effective compound for sepsis treatment.Supporting InformationThe supporting information is available online at 10.1007/s11427-021-2136-y. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

The Features of Checkpoint Receptor-Ligand Interaction in Cancer and the Therapeutic Effectiveness of Their Inhibition.

To date, certain problems have been identified in cancer immunotherapy using the inhibition of immune checkpoints (ICs). Despite the excellent effect of cancer therapy in some cases when blocking the PD-L1 (programmed death-ligand 1) ligand and the immune cell receptors PD-1 (programmed cell death protein 1) and CTLA4 (cytotoxic T-lymphocyte-associated protein 4) with antibodies, the proportion of patients responding to such therapy is still far from desirable. This situation has stimulated the exploration of additional receptors and ligands as targets for immunotherapy. In our article, based on the analysis of the available data, the TIM-3 (T-cell immunoglobulin and mucin domain-3), LAG-3 (lymphocyte-activation gene 3), TIGIT (T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains), VISTA (V-domain Ig suppressor of T-cell activation), and BTLA (B- and T-lymphocyte attenuator) receptors and their ligands are comprehensively considered. Data on the relationship between receptor expression and the clinical characteristics of tumors are presented and are analyzed together with the results of preclinical and clinical studies on the therapeutic efficacy of their blocking. Such a comprehensive analysis makes it possible to assess the prospects of receptors of this series as targets for anticancer therapy. The expression of the LAG-3 receptor shows the most unambiguous relationship with the clinical characteristics of cancer. Its inhibition is the most effective of the analyzed series in terms of the antitumor response. The expression of TIGIT and BTLA correlates well with clinical characteristics and demonstrates antitumor efficacy in preclinical and clinical studies, which indicates their high promise as targets for anticancer therapy. At the same time, the relationship of VISTA and TIM-3 expression with the clinical characteristics of the tumor is contradictory, and the results on the antitumor effectiveness of their inhibition are inconsistent.

Clinical Significance of BTLA and HVEM Expression on Circulating CD4+ T and CD8+ T Cells in Chronic Hepatitis B Virus Infection.

In this study, B and T lymphocyte attenuator (BTLA) and herpesvirus entry mediator (HVEM) expression on the surface of circulating CD4+ T and CD8+ T cells of patients with chronic hepatitis B (CHB) was investigated to explore their relationship with hepatitis B virus (HBV) clinical parameters. Both BTLA and HVEM were significantly upregulated on CD4+ T and CD8+ T cells of CHB patients compared with healthy controls (p < 0.01). Intriguingly, in CHB patients, the percentage of BTLA expression was positively correlated with that of HVEM (CD4+ T cells: r = 0.5461, p < 0.001 and CD8+ T cells: r = 0.4206, p < 0.01). Moreover, the percentage of BTLA expression was positively correlated with the levels of aspartate aminotransferase (AST) (CD4+ T cells: r = 0.3136, p < 0.05 and CD8+ T cells: r = 0.3159, p < 0.05) and alanine aminotransaminase (ALT) (CD4+ T cells: r = 0.3177, p < 0.05 and CD8+ T cells: r = 0.3311, p < 0.05). At the same time, the percentage of HVEM expression was also positively correlated with AST levels (CD4+ T cells: r = 0.3721, p < 0.05 and CD8+ T cells: r = 0.3325, p < 0.05) and ALT (CD4+ T cells: r = 0.3689, p < 0.05 and CD8+ T cells: r = 0.3476, p < 0.05). However, the percentage of BTLA and HVEM expression did not show significant relevance to HBV viral load. Further study demonstrated that BTLA inhibitory signaling could significantly inhibit T cell proliferation, activation, and cytokine production under optimal T cell receptor signaling (p < 0.05). Thereby, our findings indicate that the increased BTLA and HVEM expression on the surface of CD4+ and CD8+ T cells might represent a certain clinical significance and be involved in CHB progression during T cell exhaustion.

High BTLA Expression Likely Contributes to Contraction of the Regulatory T Cell Subset in Lupus Disease.

B and T lymphocyte attenuator (BTLA) is a co-inhibitory receptor that is expressed by lymphoid cells and regulates the immune response. Consistent with an inhibitory role for BTLA, the disease is exacerbated in BTLA-deficient lupus mice. We recently demonstrated that the BTLA pathway is altered in CD4+ T cells from lupus patients. In the present work, we aimed at delineating the expression pattern of BTLA on CD4+ T cell subsets suspected to play a key role in lupus pathogenesis, such as circulating follicular helper T cells (cTFH) and regulatory T cells (Tregs). We did not detect significant ex vivo variations of BTLA expression on total CD4+ T cells (naive and memory), cTFH or TFH subsets between lupus patients and healthy controls. However, we interestingly observed that BTLA expression is significantly increased on activated Tregs, but not resting Tregs, from lupus patients, especially those displaying an active disease. Moreover, it correlates with the diminution of the Tregs frequency observed in these patients. We also showed that both BTLA mRNA and protein expression remain low after TCR stimulation of activated Tregs sorted from healthy donors and evidenced a similar dynamic of BTLA and HVEM expression profile by human Tregs and effector CD4+ T cells upon T cell activation than the one previously described in mice. Finally, we observed that the HVEM/BTLA ratio is significantly lower in Tregs from lupus patients compared to healthy controls, whereas ex vivo effector CD4+ T cells express higher BTLA levels. Our data suggest that an altered expression of BTLA and HVEM could be involved in an impaired regulation of autoreactive T cells in lupus. These results provide a better understanding of the BTLA involvement in lupus pathogenesis and confirm that BTLA should be considered as an interesting target for the development of new therapeutic strategies.

BTLA Expression in CLL: Epigenetic Regulation and Impact on CLL B Cell Proliferation and Ability to IL-4 Production.

In our previous study, while chronic lymphocytic leukemia (CLL) cases showed higher levels of B and T lymphocyte attenuator (BTLA) mRNA compared to controls, lower BTLA protein expression was observed in cases compared to controls. Hence we hypothesize that micro RNA (miR) 155-5p regulates BTLA expression in CLL. In line with earlier data, expression of BTLA mRNA and miR-155-5p is elevated in CLL (p = 0.034 and p = 0.0006, respectively) as well as in MEC-1 cell line (p = 0.009 and 0.016, respectively). Inhibition of miR-155-5p partially restored BTLA protein expression in CLL patients (p = 0.01) and in MEC-1 cell lines (p = 0.058). Additionally, we aimed to evaluate the significance of BTLA deficiency in CLL cells on proliferation and IL-4 production of B cells. We found that secretion of IL-4 is not dependent on BTLA expression, since fractions of BTLA positive and BTLA negative B cells expressing intracellular IL-4 were similar in CLL patients and controls. We demonstrated that in controls the fraction of proliferating cells is lower in BTLA positive than in BTLA negative B cells (p = 0.059), which was not observed in CLL. However, the frequency of BTLA positive Ki67+ B cells in CLL was higher compared to corresponding cells from controls (p = 0.055) while there were no differences between the examined groups regarding frequency of BTLA negative Ki67+ B cells. Our studies suggest that miR-155-5p is involved in BTLA deficiency, affecting proliferation of CLL B cells, which may be one of the mechanisms responsible for CLL pathogenesis.

The prospective prognostic value of the immune checkpoint BTLA expression in adult acute myeloid leukemia patients

BackgroundOne of the crucial functions of the immune system is to prevent tumorigenesis, yet cancer occurs when malignant cells manage to evade immune surveillance via multiple strategies. Accordingly, this study aimed at assessing the potential significance of the novel immune checkpoint B and T lymphocyte attenuator (BTLA) as a prognostic marker in acute myeloid leukemia (AML), in addition to how it relates to response to treatment and patients’ survival. Thus, mRNA expression of BTLA was investigated on peripheral blood in 60 AML patients and 15 healthy controls.ResultsBTLA expression was found to be significantly elevated (p = 0.024) in the tested AML cases in comparison with healthy controls. Moreover, BTLA was over-expressed in the CD13, CD33, and HLA-DR positive cases as compared to their negative counterparts (p = 0.003; p < 0.001, and p = 0.001, respectively), and cases showing BTLA over-expression had significantly poorer overall survival times (p = 0.001) as confirmed by Kaplan–Meier survival analysis.ConclusionThese observations suggest that BTLA over-expression may be associated with reduced immunity against tumors and could be recommended as a promising biomarker for unfavorable prognosis in AML.

BTLA-HVEM Couple in Health and Diseases: Insights for Immunotherapy in Lung Cancer.

Lung cancer is the leading cause of cancer deaths worldwide. Immunotherapies (IT) have been rapidly approved for lung cancer treatment after the spectacular results in melanoma. Responses to the currently used checkpoint inhibitors are strikingly good especially in metastatic diseases. However, durable responses are observed in only 25% of cases. Consequently, there is an urgent need for new immunotherapy targets. Among the multiple checkpoints involved in the tumor immune escape, the BTLA-HVEM couple appears to be a promising target. BTLA (B- and T- Lymphocyte Attenuator) is a co-inhibitory receptor mainly expressed by B and T cells, repressing the activation signal transduction. BTLA shares similarities with other immune checkpoints such as PD-1 and CTLA-4 which are the targets of the currently used immunotherapies. Furthermore, BTLA expression points out terminally exhausted and dysfunctional lymphocytes, and correlates with lung cancer progression. The ligand of BTLA is HVEM (Herpes Virus Entry Mediator) which belongs to the TNF receptor family. Often described as a molecular switch, HVEM is constitutively expressed by many cells, including cells from tumor and healthy tissues. In addition, HVEM seems to be involved in tumor immuno-evasion, especially in lung tumors lacking PD-L1 expression. Here, we propose to review the role of BTLA-HVEM in immuno-escape in order to highlight its potential for designing new immunotherapies.

Simultaneous transduction of dendritic cells with A20 and BTLA genes stimulates the development of stable and efficient tolerogenic dendritic cells and induces regulatory T cells

BackgroundThis study investigated the potential of simultaneous overexpression of A20 and B- and T-lymphocyte attenuator (BTLA) genes in dendritic cells (DCs) to develop a tolerogenic phenotype in DCs and investigate their capabilities for induction of immunosuppression. MethodsPlasmid vectors were designed harboring A20, BTLA, and A20 + BTLA genes and were transfected to HEK 293T cells to produce lentiviruses. DCs were transduced by the gene carrying viruses and evaluated for the surface expression of MHCII, CD40, and CD86 molecules by flow-cytometry. The mRNA expression of A20, BTLA, and CCR7 were determined. Mixed-lymphocyte reaction was conducted to evaluate the T cell stimulation potency and ELISA was used to measure the production of IL-10, TGF-β, and TNF-α. The potential of DCs for migration to lymph nodes and Treg induction were assessed by in vivo experiments. ResultsTransduction of DCs resulted in significantly decreased surface expression of CD40 and CD86 co-stimulators and upregulated A20, BTLA, and CCR7 mRNA expression. The IL-10 and TGF-β levels were enhanced significantly in the supernatant of LPS-treated DCs transduced with A20 + BTLA-containing virus group relative to the DCs transduced with pCDH vectors. DCs transduced with A20 + BTLA harboring vectors had higher migratory potential to mouse lymph nodes and caused the development of higher numbers of Treg cells compared with the DCs transduced with pCDH vectors. ConclusionsSimultaneous overexpression of A20 and BTLA genes in DCs caused development of tolerogenic DCs with a promoted potential in induction of Treg cells, accompanied by remarkable stability after inflammatory stimulation. All these offer a promising potential of such DCs in treating autoimmune and inflammatory disorders.

Intronic SNP - rs9288953 Of Suppressor Molecule Btla Confers Susceptibility To B-Cell Chronic Lymphocytic Leukemia

The contribution of the immune system to the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) have been receiving growing attention. The increase of cancer risk in immunodeficient individuals has been documented in population and experimental studies (Gurlich et al 2007; Swann et al 2007, Smyth et al 2006). A new member of co-inhibitory molecules B - and T-lymphocyte attenuator (BTLA) that inhibits lymphocyte activation could influence cancer susceptibility (Watanabe et al 2003). A high expression of BTLA was observed on malignant B cells from chronic lymphocytic leukemia/small lymphocytic leukemia (M'Hidi et al. 2009). As BTLA synthesis depends on the rate of gene transcription and/or translation, polymorphism in the corresponding gene might result in abnormal expression and function.The aim of this studywas to evaluate possible associations between selected on the basis of literature (Fu et al 2009) single nucleotide polymorphisms (SNPs) existing within exons and introns of BTLA gene: rs9288952A>G, rs1844089G>A, rs2705535C>T and 9288953C>T and susceptibility to B-CLL.Genotyping was done using allelic discrimination method with the TaqMan SNP Genotyping Assays in 300 B-CLL patients (pts.) (203 pts. from Department of Hematology, Medical University Lodz, Poland and 97 from Department of Clinical Immunology and Immunotherapy, Medical University of Lublin, Poland (138 female/162 male) and in 417 healthy individuals (168 female/243 male) originating from the same region of Poland.The significant differences in distribution of alleles and genotypes was observed for intronic SNP -rs9288953. The frequency of T allele and T alleles carriers (CT+TT genotypes) were higher in patients than in healthy controls as compare to C allele and CC genotypes (0.458 vs. 0.375, p=0.0016; OR:1.41, 95%CI - 1.13-1.75 and 0.677 vs. 0.606, p=0.0022; OR:1.67, 95%CI- 1.16-1.2.40, respectively). None of other investigated SNPs have shown different frequency of alleles or genotypes in group of patients and controls.The haplotype analysis of rs9288952A>G, rs1844089G>A, rs2705535C>T and 9288953C>T showed that the most common haplotypes rs9288952A>G[A]/rs1844089G>A[G]/rs2705535C>T[C]/ 9288953C>T[C] (named as AGCC) and rs9288952A>G[A]/rs1844089G>A[G]/rs2705535C>T[C]/ 9288953C>T[T] (named as AGCT) which were differentiate only by variants of 9288953C>T SNP were completely contrary associated with disease susceptibility. Haplotype AGCC is associated with decreased risk of B-CLL (OR: 0.72, 95%CI-0.59-0.90 p with Bonferroni correction=0.01), while AGCT with increased risk of disease (OR: 1.42, 95%CI-1.14-1.76 p with Bonferroni correction=0.005)Our data indicated for the first time the possible role of intronic polymorphism 9288953C>T in gene encoding suppressor molecule BTLA in susceptibility to B-CLL. ReferencesFu Z, Li D, Jiang W, Wang L, Zhang J, Xu F, Pang D, Li D. Association of BTLA gene polymorphisms with the risk of malignant breast cancer in Chinese women of Heilongjiang Province. Breast Cancer Res Treat. 2010 Feb;120(1):195-202.Grulich AE, van Leeuwen MT, Falster MO, Vajdic CM. Incidence of cancers in people with HIV/AIDS compared with immunosuppressed transplant recipients: a meta-analysis. Lancet. 2007 Jul 7;370(9581):59-67.M'Hidi H, Thibult ML, Chetaille B, Rey F, Bouadallah R, Nicollas R, Olive D, Xerri L. High expression of the inhibitory receptor BTLA in T-follicular helper cells and in B-cell small lymphocytic lymphoma/chronic lymphocytic leukemia.Am J Clin Pathol. 2009 Oct;132(4):589-96.Smyth MJ, Dunn GP, Schreiber RD. Cancer immunosurveillance and immunoediting: the roles of immunity in suppressing tumor development and shaping tumor immunogenicity. Adv Immunol. 2006;90:1-50.Swann JB, Smyth MJ. Immune surveillance of tumors. J Clin Invest. 2007 May;117(5):1137-46.Watanabe N, Gavrieli M, Sedy JR, Yang J, Fallarino F, Loftin SK, Hurchla MA, Zimmerman N, Sim J, Zang X, Murphy TL, Russell JH, Allison JP, Murphy KM. BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1.Nat Immunol. 2003 Jul;4(7):670-9. Disclosures:No relevant conflicts of interest to declare.

Genetic signature of CTLA-4, BTLA, TIM-3 and LAG-3 molecular expression in colorectal cancer patients: Implications in diagnosis and survival outcomes

ObjectiveAccumulating evidences suggest that immune checkpoints (ICs) inhibit immune response against cancerous cells and promote tumor cell survival. Up-regulation of ICs in tumor microenvironment is reported in patients with colorectal cancer (CRC). Thus, evaluating the peripheral blood expression of ICs may be used as non-invasive biomarkers for diagnosis and prognosis of CRC. MethodsThis study included 60 primary and treatment naïve CRC patients along with 15 age and sex matched healthy volunteers as a control group. Total RNA was extracted from peripheral blood samples and gene expression of cytotoxic T lymphocyte antigen-4 (CTLA-4), B and T lymphocyte attenuator (BTLA), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and Lymphocyte activation gene-3 (LAG-3) was measured by quantitative real time polymerase chain reaction (qRT-PCR). All patients were followed for 12 months to correlate the measured ICs to patients’ survival. ResultsThe gene expression of CTLA-4, BTLA, TIM-3 and LAG-3 was significantly up-regulated in CRC patients compared to the control group (p < 0.001). Individually, CTLA-4 and BTLA showed 85% sensitivity in discriminating CRC patients from control group (p < 0.001). On the other hand, TIM-3 and LAG-3 expression showed higher sensitivity (93%) for diagnosis of CRC (p < 0.001). Conversely, CTLA-4 or BTLA strongly predicted CRC patients’ survival (p < 0.001) compared to TIM-3 (p = 0.018) or LAG-3 (p = 0.035). CTLA-4, BTLA, TIM-3 and LAG-3 were independent prognostic factors of survival after adjustment for age and gender. ConclusionThe current study provided evidence that blood gene expression of ICs was up-regulated in CRC patients and associated with cancer stage and patients’ survival, which highlights the diagnostic and prognostic values of ICs expression in CRC. Further investigations and validations in larger cohorts are required.

Serum levels of soluble B and T lymphocyte attenuator predict overall survival in patients undergoing immune checkpoint inhibitor therapy for solid malignancies.

Therapy with immune checkpoint inhibitors (ICIs) can lead to durable tumor control in patients with various advanced stage malignancies. However, this is not the case for all patients, leading to an ongoing search for biomarkers predicting response and outcome to ICI. The B and T lymphocyte attenuator (BTLA) is an immune checkpoint expressed on immune cells that was shown to modulate therapeutic responses. Here, we evaluate circulating levels of its soluble form, soluble B and T lymphocyte attenuator (sBTLA), as a biomarker for the prediction of treatment response and outcome to ICI therapy. Serum levels of sBTLA were analyzed by multiplex immunoassay in n=84 patients receiving ICI therapy for solid malignancies and 32 healthy controls. BTLA expression was evaluated on peripheral blood mononuclear cells in a subset of patients (n=6) using multicolor flow cytometry. Baseline sBTLA serum levels were significantly higher in cancer patients compared to healthy controls. Importantly, circulating sBTLA levels were an independent prognostic factor for overall survival (OS). As such, patients with initial sBTLA levels above the calculated prognostic cutoff value (311.64 pg/mL) had a median OS of only 138 days compared to 526 for patients with sBTLA levels below this value (P= .001). Uni- and multivariate Cox regression analyses confirmed the prognostic role of sBTLA in the context of ICI therapy. Finally, we observed a significant correlation between sBTLA levels and the frequency of CD3 + CD8 + BTLA+ T cells in peripheral blood. Thus, our data suggest that circulating sBTLA could represent a noninvasive biomarker to predict outcome to ICI therapy, helping to select eligible therapy candidates.

Up-regulation of BTLA expression in myeloid dendritic cells associated with the treatment outcome of neonatal sepsis

Dentritic cells (DCs) dysfunction has been verified detrimental for sepsis and B and T lymphocyte attenuator (BTLA) is an immune-regulatory receptor shown to be associated with DCs dysfunction. However, the role of BTLA expression in myeloid DCs (mDCs) in neonatal sepsis is unknown. In the current study, we found BTLA-expressing mDCs were elevated in neonates with sepsis and the BTLA expression level in mDCs was positively correlated to the severity of sepsis. The presence of BTLA negatively regulated the phagocytosis capacity and bactericidal ability of mDCs as well as the maturation markers expression of mDCs. Our data also showed BTLA+mDCs shifted into an anti-inflammatory phenotype with decreased expression of IL-6, TNF-α and IL-12, but increased IL-10. in addition, we found BTLA expression indeedly altered the mDCs allo-stimulatory capacity. Therefore, BTLA expression in mDCs could be a useful predictive marker for neonatal sepsis and targeting BTLA expression in mDCs may be a new therapeutic strategy.

B and T lymphocyte attenuator regulates autophagy in mycobacterial infection via the AKT/mTOR signal pathway

The survivability of Mycobacterium tuberculosis (M.tb) in macrophages in granuloma is a predominant cause for tuberculosis (TB) infection and recurrence. However, the mechanism of mycobacterial clearance in macrophages still needs further study. Here, we explored a novel role of B and T lymphocyte Attenuator (BTLA) in macrophage-mediated host defense against mycobacterial infection. We found that the surface expression of BTLA was increased in CD14+ monocytes from active TB patients. The mRNA levels of BTLA were induced in human and mice monocytes/macrophages during Mycobacterium bovis BCG or M.tb H37Rv infection, as well as spleen and lung of H37Rv-infected mice. Furthermore, silencing of BTLA promoted the intracellular survival of BCG and H37Rv by suppressing the autophagy in macrophages but not effecting phagocytosis, reactive oxygen species (ROS) and apoptosis. Silence of BTLA reduced bacterial-autophagosome and bacterial-lysosome colocalization. Moreover, BTLA inhibited AKT and mTOR signaling substrates S6K and 4EBP1 phosphorylation in BCG and H37Rv infected macrophages, and BTLA-mediated AKT-mTOR signaling and intracellular BCG survival were reversed by PI3K inhibitors in macrophages. Finally, treatment with BTLA agonist ameliorated lung pathology and promoted autophagy and mycobacterial clearance during mycobacterial infection in vivo. These results demonstrate that BTLA promotes host defense against mycobacteria by enhancing autophagy, which may provide potential therapeutic interventions against tuberculosis.